Ng-monomethyl-l-arginine hydrochloride derivatives and their use in the treatment of septic shock

ABSTRACT

Crystalline N G  -monomethyl-L-arginine hydrochloride is disclosed. At least three distinguishable isomorphic forms are present. The solid salt can be made by dissolving N G  -monomethyl-L-arginine in hydrochloric acid and crystallising out N G  -monomethyl-L-arginine hydrochloride. The crystallising step requires several months at low temperature. However, the process can be facilitated by seeding with crystals of N G  -monomethyl-L-arginine hydrochloride. Alternatively, crystalline N G  -monomethyl-L-arginine hydrochloride can be prepared by dissolving a salt of N G  -monomethyl-L-arginine other than the hydrochloride salt with hydrochloric acid and removing the original salt forming ion by crystallising out the hydrochloride salt.

This is a division of application Ser. No. 08/862,468, filed May 23,1997, now U.S. Pat. No. 5,767,312 which is a Continuation of Ser. No.08/461,163, filed Jun. 5, 1995, now abandoned; which is a continuationof Ser. No. 08/374,636, filed Jan. 23, 1995, now abandoned.

The present invention relates to novel crystalline salts of (S)-N5-imino(methyl amino)methyl!ornithine, pharmaceutical compositionscontaining such salts and their use in medicine, more particularly thetreatment and/or prophylaxis of septic shock especially the hypotensionassociated therewith.

International patent application WO91/04024 (U.S. Pat. No. 5,028,627)describes the use of an N^(G) substituted arginine or an N^(G) -N^(G)-disubstituted arginine to treat hypotension. In particular, this patentapplication describes the use of N^(G) -monomethyl-L-arginine (alsoknown as (S)-N⁵ imino(methylamino)methyl!ornithine or L-NMMA) tocounteract the production of nitric oxide in induced hypotension andseptic shock. Nitric oxide is a potent vasodilator and cytotoxic agentthat is normally produced in the endothelium as an endogenous regulatorof vascular tone and in macrophages as part of the host defencemechanism. Inappropriate increase in nitric oxide synthesis leads toexaggeration in these actions, so as to cause sustained and pronouncedvasodilation, leading to hypofusion of various vital organisms.Furthermore, the substantial increase in the synthesis of nitric oxidein the number of cells leading to cytotoxicity and direct tissue damage,especially to the vascular endothelium.

Kilbourn et al (Proc. Natl. Acad. Sci. U.S.A., 87, 3629, 1990) reportthe reaction of the flavianate salt of LNMMA with Dowex 1(OH⁻) andtitrating the resulting free base of L-NMMA to pH 7.2 with hydrochloricacid. The hydrochloride was not isolated from solution.

We have now found that hydrochloric of L-NMMA may be obtained as a puresalt, for example in a crystalline form, which has considerable physicaladvantages, for example in terms of stability. Initial attempts toprepare the hydrochloride in crystalline form gave rise to an amorphousglass which did not crystallise. Crystals of the hydrochloride wereunexpectedly obtained when the amorphous glass was left in the presenceof ethanol for several months.

Accordingly, the present invention provides the hydrochloride salt of(S)-N⁵ imino(methylamino)methyl!ornithine as a substantially pure saltfor example in a solid form and more specifically in a crystalline form.This hydrochloride salt of L-NMMA is anhydrous and not hydroscopic.

Preferably the salt is at least 70%, more preferably at least 90% pureand most preferably greater than 95% pure.

The salt exists in at least three distinguishable isomorphic form (A,Band C) as identified by X-ray diffraction analysis and DifferentialScanning Calorimetry (DSC), and the present invention is intended toinclude each isomorphic form individually or a mixture of two or moreisomorphic forms.

Hygroscopicity studies have shown Form A to deliquesce at 65% humidity,whilst Form B is considerably less hygroscopic. DSC shows Form A to bethe more thermodynamically stable. DSC has also been used to estimatethe melting points of Forms A and B as 219° C. and ca.205° respectively.

The present invention also provides a process for preparing crystallineL-NMMA hydrochloride which process comprises reacting L-NMMA withhydrochloric acid and crystallising out the hydrochloride. Preferablythe molar ratio of L-NMMA to acid is from 1:1 to 1:5 and, in particular,approximately 1 to 1. The reaction is suitably carried out by dissolvingthe L-NMMA in a solution of hydrochloric acid (preferably between 0.5molar and 5 molar and conveniently 2 molar, at a non-extremetemperature, for example between 10° and 80° C., and conveniently atroom temperature. The resulting solution is preferably evaporated (forexample at a raised temperature, i.e. between 35° and 60°, under reducedpressure). The residue is then crystallised from a suitable solvent, forexample by dissolving the residue in a minimum of hot ethanol,conveniently at boiling point, and water. It has been found that seedingthe solution containing the hydrochloride salt of L-NMMA assists itscrystallisation. Without seeding, the crystallation process may takeseveral months.

The present invention further provides a process for producing L-NMMAhydrochloride which process comprises reacting a salt of L-NMMA otherthan the hydrochloride with hydrochloric acid and removing the originalsalt forming ion. Conveniently the reaction is carried out by dissolvingthe acetate salt of L-NMMA, in aqueous hydrochloric acid, at aconcentration between 0.5 and 5 molar conveniently 2 molar. The solutionis then evaporated (for example at an elevated temperature, i.e. between30° and 70°, conveniently 60°, under reduced pressure) The residue isthen dissolved in a suitable solvent, for example aqueous ethanol andcooled. Again, seeding the cooled solution with crystals of thehydrochloric salt of L-NMMA assists the crystallisation procedure.

Whilst it may be possible for the hydrochloride of L-NMMA to beadministered as the raw chemical, it is preferable to present it as apharmaceutical formulation. According to a further aspect, the presentinvention provides a pharmaceutical formulation comprising thehydrochloride of L-NMMA (the "active ingredient") together with one ormore pharmaceutically acceptable carriers therefor and optionally one ormore other therapeutic ingredients. The carrier(s) must be "acceptable"in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous andintraarticular), rectal and topical (including dermal, buccal,sublingual and intraocular) administration although the most suitableroute may depend upon, for example, the condition and disorder of therecipient. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing into association theactive ingredient with the carrier which constitutes one or moreaccessory ingredients. In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both and then,if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tables may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example, saline, water-for-injection,immediately prior to use or may be stored as a solution ready forinjection. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Formulations for rectal administration may be presented as a suppositorywith the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basesuch as gelatin and glycerin or sucrose and acacia.

Preferably the salt will be administered as a solution in water bufferedto its own pKa.

Preferably unit dosage formulations are those containing an effectivedose, as hereinbelow recited, or an appropriate fraction thereof, of theactive ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The compounds of the invention may be administered orally or viainjection at a dose of from 1 to 100 mg/kg per day and preferably 3 to50 mg/kg per day. Doses of above 3 mg/kg per day may preferably be givenin a series of smaller doses over a prolonged period, i.e. by infusionover several hours. The dose range for adult humans is generally from 70mg to 7 g/day and preferably 200 mg to 3.5 g/day. Tablets or other formsof presentation provided in discrete units may conveniently contain anamount of compound of the invention which is effective at such dosage oras a multiple of the same, for instance, units containing 70 mg to 500mg, usually around 100 mg to 300 mg.

The compounds of the invention are most suitably administered orally orby injection (intravenous or subcutaneous) and preferably by injection.The precise amount of compound administered to a patient will be theresponsibility of the attendant physician. However the dose employedwill depend on a number of factors, including the age and sex of thepatient, the precise disorder being treated, and its severity. Also theroute of administration may vary depending on the condition and itsseverity.

As mentioned hereinbefore N^(G) -monomethyl-L-arginine hydrochloride maybe of use in the treatment and/or prophylaxis of septic shock.Accordingly, in a further aspect of the present invention there isprovided N^(G) -monomethyl-L-arginine hydrochloride for use in themanufacture of a medicament for the treatment and/or prophylaxis ofseptic shock. A yet further aspect of the present invention provides amethod of treatment or prophylaxis of septic shock which comprises theadministration of a therapeutically effective amount of N^(G)-monomethyl-L-arginine hydrochloride.

The invention will now be described by way of example only.

EXAMPLE 1

Preparation of (S)-N5- Imino(methylamino)methyl!ornithine hydrochloride(L-NMMA hydrochloride)

A mixture of ornithine hydrochloride (16.86 g, 100 mMol),N,S-dimethylthiouronium iodide (34.8 g, 150 mMol) and 2M aqueous sodiumhydroxide (100 ml) was stirred at 100° C. for five hours. The solutionwas then cooled, adjusted to pH3 with 2M aqueous hydrochloric acid, andapplied to a column of Dowex 50W-X8 (H+) resin (200 ml wet bed volume).The column was washed with water until the eluate was neutral and theneluted with 0.5M aqueous ammonium hydroxide; fractions of approximately15 ml were taken and were monitored by the silica gel with visualisationby ninydrin. Fractions 26-45 were combined and evaporated at 45° C.under reduced pressure to give a colourless resin (9.5 g) which wasdissolved in 2M aqueous hydrochloric acid and the resulting solution wasevaporated at 50° C. under reduced pressure. The amorphous residue wastreated with hot ethanol (108 ml) and the mixture was stirredvigorously, at the boiling point, during the dropwise addition of water(4 ml). The residue gradually dissolved and on seeding with a fewcrystals of the hydrochloride salt began to crystallise. The mixture wascooled and then stood at 4° C. for two hours to complete thecrystallisation. The product was removed by filtration, washed withethanol and dried in a vacuum dessicator to give pure L-NMMAhydrochloride (9.6 g) as an anhydrous colourless crystalline solid,homogeneous by tlc and hpic and with ¹ H nmr and mass spectrumconsistent with the proposed structure.

EXAMPLE 2

Preparation of L-NMMA hydrochloride from the acetate monohydrate

L-NMMA acetate monohydrate (186 g) was dissolved in 2M aqueoushydrochloride acid (350 ml) and the solution was evaporated at 60° C.under reduced pressure. The residue was then dissolved in water (ca 150ml) and evaporated under reduced pressure; the redissolution andevaporation process was then repeated twice. The residue was thendissolved in warm water (25 ml) with the addition of ethanol (25 ml) toaid mobility. The still warm solution was stirred, treated with ethanol(1200 ml), seeded with a few crystals and stirring was continued at roomtemperature for 5 hours. The mixture was kept overnight at 4° C. and thecrystalline solid was filtered, washed with ethanol and dried in avacuum desiccator to give pure L-NMMA hydrochloride (108 g) identical inall respects with the material described above.

EXAMPLE 3

A sample of amorphous hydrochloride, prepared as in Example 1 butwithout seeding, crystallised after standing under ethanol for aboutfive months at 4° C.; this material was used for "seeding" thepreparations of Examples 1 and 2.

Samples of seed crystals of L-NMMA hydrochloride are available, onrequest, from the School of Chemistry, the University of Birmingham,Birmingham B15 2TT.

I claim:
 1. A sterile pharmaceutical composition for parenteraladministration which comprises N^(G) -monomethyl-L-argininehydrochloride with a pharmaceutically acceptable carrier.
 2. A sterilepharmaceutical composition for parenteral administration which comprisesN^(G) -monomethyl-L-arginine hydrochloride buffered to its own pKa in apharmaceutically acceptable carrier.
 3. An ampoule or vial containing asterile aqueous solution for parenteral administration of N^(G)-monomethyl-L-arginine hydrochloride.
 4. An ampoule or vial containinglyophilized N^(G) -monomethyl-L-arginine hydrochloride.
 5. A method oftreating septic shock comprising parenterally administering to a patientin need of same a therapeutically effective amount of a sterilepharmaceutical composition which comprises N^(G) -monomethyl-L-argininehydrochloride in a pharmaceutically acceptable carrier.
 6. The method ofclaim 5 in which the composition is administered by injection at a doseof 1 to 100 mg/kg per day.
 7. The method of claim 6 in which thecomposition is administered by injection at a dose of 3 to 50 mg/kg perday.